Materials and methods for detection of Oxalobactor formigenes

ABSTRACT

The subject invention concerns the novel use of formyl-CoA transferase enzyme together with oxalyl-CoA decarboxylase enzyme for the detection and measurement of oxalate in biological samples. The use of the enzyme system according to the subject invention results in the conversion of oxalate into carbon dioxide and formate. Because the production of formate is directly correlated to the concentration of oxalate present in a sample, the determination of the resulting formate concentration provides an accurate, sensitive and rapid means for detecting even low levels of oxalate. The subject invention further concerns the cloning, sequencing and expression of the genes that encode the formyl-CoA transferase enzyme and the oxalyl-CoA decarboxylase enzyme of Oxalobacter formigenes. The subject invention also concerns methods for detecting the presence of Oxalobacter formigenes organisms in a sample, and the polynucleotide probes and primers used in the detection method.

This invention was made with government support under NationalInstitutes of Heath Grant No. DK 20586. The government has certainrights in this invention.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of patent application Ser.No. 08/717,587, filed Sep. 27, 1996 now U.S. Pat. No. 5,912,125; whichis a continuation-in-part of patent application Ser. No. 08/493,197,filed June 20, 1995 now U.S. Pat. No. 5,837,833; which is acontinuation-in-part of patent application Ser. No. 08/262,424, filedJun. 20, 1994, now U.S. Pat. No. 5,604,111.

FIELD OF INVENTION

The present invention relates to novel assay methods and devices fordetermining the presence or concentration of oxalate in a sample;Oxalobacter genes encoding enzymes required for the catabolism ofoxalate; and materials and methods for detecting and identifyingOxalobacter formigenes in a sample.

BACKGROUND OF THE INVENTION

Oxalic acid (Oxalate) is a highly toxic natural by-product of catabolismin vertebrate animals and many consumable plants. Unfortunately, asignificant portion of humans are unable to properly metabolizingoxalate, a condition which may result in the formation of kidney stonesin those persons. It is estimated that 70% of all kidney stones arecomposed of some amount of oxalate. Approximately 12 percent of the U.S.population will suffer from a kidney stone at some time in their lives,and the incidence is rising not only in the United States, but also inSweden and Japan (Curhan, 1993). Moreover, although a healthy personbreaks down or excretes sufficient quantities of oxalate to avoidexcessive accumulation of oxalate in the tissues, a number of diseasestates are known to be associated with malfunctions of oxalatemetabolism, including pyridoxine deficiency, renal failure and primaryhyperoxaluria, a metabolic genetic disorder that results in theexcessive deposition of oxalate in the kidneys.

Persons suffering from and at risk for developing kidney stones, as wellas patients with lipid malabsorption problems (e.g., sprue, pancreaticinsufficiency, inflammatory intestinal disease, bowel resection, etc.),tend to have elevated levels of urinary oxalate, a fact that has beenexploited as a means for identifying individuals at risk. While elevatedlevels of oxalate may be present in urine, detecting elevated levels ofoxalate in serum has not been routine due to the difficulty in detectingthe low levels of oxalate present in serum.

Most previous methods for measuring oxalate in a biological sample firstrequire the isolation of the oxalate by precipitation, solventextraction, or an ion-exchange absorption (Hodgkinson, 1970).Quantitation of the isolated oxalate may be determined by any one ofseveral methods including colorimetry, fluorometry, gas-liquidchromatography or isotope dilution techniques. Because many of theoxalate isolation techniques used in these analytical methods are notquantitative, it is normally necessary to correct for the low recoveryof oxalate by adding a ¹⁴ C-labeled oxalic acid internal standard, whichfurther complicates the analytical method. All these methods arelaborious, and consequently expensive because of the amount of skilledlaboratory technician time which must be employed. In addition,isolation of the oxalate may require relatively large sample volumes forstarting material.

Recently, several advances in the detection and quantitation of oxalatehave been made through the use of (a) oxalate degrading enzymes and (b)high performance liquid chromatography. One commercially-availableenzymatic test (Sigma Chemical Company, St. Louis, Mo.) employs oxalateoxidase to oxidize oxalate to carbon dioxide and hydrogen peroxide. Thehydrogen peroxide produced can then be measured colorimetrically in asecond enzymatic reaction in the presence of peroxidase.

In another enzymatic method for measuring oxalate, oxalate decarboxylaseis used to convert oxalate to carbon dioxide and formate. The resultantcarbon dioxide can be measured manometrically, by the pH change in acarbon dioxide trapping buffer or by the color change in a pH indicatorbuffer. Whatever method of carbon dioxide assay is adopted, the timerequired for diffusion and equilibration of carbon dioxide is muchlonger than is desirable for a rapid analytical method.

Alternatively, the formate produced by the action of oxalatedecarboxylase can be assayed with formate dehydrogenase in an NAD/NADHcoupled reaction, as described in Costello, 1976 and Yriberri, 1980.This method is both cumbersome and time-consuming because oxalatedecarboxylase and formate dehydrogenase differ in their optimum pHrequirements, thus necessitating a pH adjustment during the analysis.

Another commercially available enzymatic test (Boehringer Mannheim)cleaves oxalate to formate and carbon dioxide, then oxidizes the formateto bicarbonate by NAD in the presence of the enzyme formatedehydrogenase. The amount of NADH is determined by means of itsabsorbance at 334, 340, or 365 nm. Another test ("STONE RISK" by MissionPharmacal) measures oxalate as a part of a battery of tests for kidneystones.

Oxalobacter formigenes is a recently discovered, oxalate-degradingobligately anaerobic bacterium residing primarily in the intestines ofvertebrate animals, including man (Allison et al., 1986). Although thefirst isolates of O. formigenes were cultured from sheep rumen (Dawsonet al, 1980), additional strains have now been isolated from cecalcontents of rats, guinea pigs and pigs (Argenzio et al., 1988, Daniel etal, 1987), fecal samples from man (Allison et al., 1985), and anaerobicaquatic sediments (Smith et al., 1985). This bacterium is unique amongoxalate-degrading organisms having evolved a total dependence on oxalatemetabolism for energy (Dawson et al., 1980). Recent evidence suggeststhat Oxalobacter formigenes has an important symbiotic relationship withvertebrate hosts by regulating oxalic acid absorption in the intestineas well as oxalic acid levels in the plasma (Hatch and Freel, 1996).Studies by Jensen and Allison (1994) comparing various O. formigenesisolates revealed only limited diversity of their cellular fatty acids,proteins, and nucleic acid fragments. Based on these comparisons,strains of O. formigenes have been divided into two major subgroups. Ingeneral, group I strains have shown limited intragroup diversity, whilegroup II strains have shown greater intragroup diversity.

Special conditions are required to culture O. formigenes and theirdetection is based generally on the appearance of zones of clearance ofcalcium oxalate crystals surrounding colonies (Allison et al., 1986).Assays based on the appearance of zones of clearance of calcium-oxalatecrystals surrounding bacterial colonies (Allison et al., 1985) ordegradation of oxalate in culture media measured by calcium-chlorideprecipitation (Dawson et al., 1980) fail to confirm theoxalate-degrading bacteria as Oxalobacter.

As illustrated above, the currently existing assays for oxalate sufferfrom numerous problems, including cost, inaccuracy, reliability,complexity, and lack of sensitivity. Accordingly, it is an object of thesubject invention to provide a simple, accurate, and sensitive assay forthe detection of low levels of oxalate in a biological sample.

The current methods for culturing and identifying the presence ofOxalobacter formigenes are technically demanding and time consuming, andtherefore, are not suitable for rapid and specific identification of O.formigenes, particularly for clinical diagnostics. Accordingly, anotherobject of the subject invention is to provide a rapid, accuratepolynucleotide probe-based assay for the detection of O. formigenes.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the cloning, sequencing, and expressionof the formyl-CoA transferase (frc) and the oxalyl-CoA decarboxylase(oxc) genes of Oxalobacter formigenes, and the use of the enzymes todetect the presence of oxalate in a sample. The assay of the subjectinvention provides, for the first time, a rapid, sensitive method todetect even very low concentrations of oxalate in biological samples.Advantageously, the biological samples in which oxalate can be detectedinclude both urine and serum samples. The enzyme system used accordingto the subject invention converts oxalate to carbon dioxide and formate.In a preferred embodiment of the subject invention, the production offormate is then measured colorimetrically. This assay provides asensitive, accurate and convenient means for detecting oxalate.

A further aspect of the subject invention is the discovery of the O.formigenes genes which encode the formyl-CoA transferase and theoxalyl-CoA decarboxylase enzymes. The discovery of these genes makes itpossible to efficiently produce large quantities of pure formyl-CoAtransferase and oxalyl-CoA decarboxylase for use in the assay of thesubject invention or other appropriate application.

The subject invention further concerns a dipstick device for thedetection and quantitation of oxalate in a sample. The dipstick devicecomprising comprises the oxalyl-CoA decarboxylase and formyl-CoAtransferase enzymes of the present invention immobilized on a carriermatrix. A detectable signal is generated on the dipstick if oxalate ispresent in the sample.

The subject invention also provides a means for detecting the presenceof Oxalobacter formigenes organisms in a sample. The method of detectionprovided for herein involves polynucleotide probes which can be used toidentify Oxalobacter formigenes.

The subject invention also concerns the polynucleotide primers and theuse thereof for polymerase chain reaction (PCR) amplification ofOxalobacter formigenes nucleotide sequences. Amplified Oxalobactersequences can then be detected using the polynucleotide probes of thesubject invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the detection of varying concentrations of oxalate in asample. Colorimetric absorbance for each sample was plotted over time(minutes). Positive and negative control panels are also shown.

FIG. 2 shows the nucleotide sequence of the formyl-CoA transferase gene(SEQ ID No. 1) and the deduced amino acid sequence of the formyl-CoAtransferase polypeptide from Oxalobacter formigenes. Bolded lettersrepresent amino acid residues determined by N-terminal proteinsequencing.

FIG. 3 shows the nucleotide sequence of the oxalyl-CoA decarboxylasegene (SEQ ID No. 3) and flanking regions from Oxalobacter formigenes.The consensus ribosome-binding site lies approximately 10 bases upstream(double-underlined letters) from the putative translation initiationcodon (positions 1 to 3). A rho-independent termination sequence lies atpositions 1758 to 1790 (double-underlined letters). A putativeTPP-binding site appears between positions 1351 and 1437.

FIG. 4 shows an RFLP analysis of O. formigenes, strain OxB using probesspecific for the oxc gene encoding oxalyl-CoA decarboxylase and the frcgene encoding formyl-CoA transferase. Genomic DNA isolated from a 14 dayculture of O. formigenes strain OxB was digested with the restrictionenzyme HIND III. The digested DNA was size fractionated byelectrophoreses through 0.5% agarose gels, electroblotted to a nylonmembrane, then hybridized with either probe AP15 (SEQ ID No. 6) or probeAP34 (SEQ ID NO. 9) to detect oxc or probe AP273 (SEQ ID NO. 10) todetect frc.

FIG. 5 shows the sensitivity of detecting the oxc and frc genes in RFLPof O. formigenes strain OxB versus strain HC-1. Genomic DNA from each ofthe two strains was digested with the restriction enzyme HIND III.Two-fold serial dilutions were made of the digested DNA and sizefractionated by electrophoresis through 0.5% agarose gels (left panels).RFLP analyses were carried out as described in FIG. 4, except the nylonmembranes were hybridized with a 1: 1 mixture of probe AP15 (SEQ ID NO.6) plus probe AP273 (SEQ ID NO. 10) (right panels).

FIG. 6 shows the detection of the oxc and frc genes in various strainsof O. formigenes by RFLP analysis. RFLP was carried out as described inFIG. 5.

FIG. 7 shows PCR-based amplification of a genetic region of the oxc genein various strains of O. formigenes. Using PCR primer AP15 (SEQ ID NO.6) and primer AP22 (SEQ ID NO. 11) as PCR primers, PCR amplification wasperformed using genomic DNA isolated from each of the 12 strains of O.formigenes listed in Table 1 as template. PCR products were sizefractionated by electrophoresis through 1.2% agarose gels and observedvisually using ethidium bromide (EtBr) and UV light.

FIG. 8 shows a direct analysis of fecal samples for O. formigenes.Oxalobacter negative stool sample (A & B) was spiked with 10² (C) and10⁴ (D) cfu of OxB or 10³ (E) and 10⁴ (F) cfu of OxK per 0.1 gm. DNAfrom an unspiked O. formigenes-positive stool sample diluted 1:25 (G)and 1:50 (H).

FIG. 9 shows the identification of sequence homologies within the oxcgene expressed in representative group I and group II strains ofOxalobacter formigenes to design oligonucleotide probes. Partialsequences of 5'-end of the oxc gene generated by PRC amplification ofthe region bounded by the primer pair, AP34/AP21. A region of highhomology shared by all strains (between bp 13 and 43) was selected forthe genus-specific oligonucleotide probe, AP286, while regions of highhomology shared by only group I strains (between bp 197 and 214) orshared only by group II strains (between bp 133 and 150) were selectedfor group-specific oligonucleotide probes, HS2 and AP307, respectively.

FIGS. 10A-10B shows the detection of Oxalobacter formigenes using agenus-specific oligonucleotide probe that hybridizes to the PCR productof the oxc gene. Using the primer pair AP34/AP21, PCR amplification wasperformed using genomic template DNA isolated from 8 group I and 8 groupII strains of O. formigenes. The PCR products were size fractionated byelectrophoresis through 1.2% agarose gels and the expected 504-508 bpproduct visualized with EtBr under UV light (upper panel). The PCRproducts were transblotted to nylon membranes and Southern blotted usingthe genus -specific oligonucleotide probe, AP286 (lower panel).

FIGS. 11A-11C shows the classification of group I and group II strainsof Oxalobacter formigenes using group-specific oligonucleotide probesthat hybridize with PCR products of the oxc gene. Using the primer pairAP34/AP21, PCR amplification was performed using genomic template DNAisolated from 8 group I and 8 group II strains of O. formigenes. The PCRproducts were size fractionated by electrophoresis through 1.2% agarosegels and the expected 504-508 bp product visualized with EtBr under UVlight (upper panel). The PCR products were transblotted to nylonmembranes and Southern blotted using HS2, the group I-specific (centerpanel), or AP307, the group II-specific (lower panel), oligonucleotideprobes.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a nucleotide sequence for the formyl-CoA transferasegene (also shown in FIG. 2).

SEQ ID NO. 2 is a polypeptide encoded by SEQ ID NO. 1, which can be usedaccording to the subject invention.

SEQ ID NO. 3 is the nucleotide sequence for the oxalyl-CoA decarboxylasegene (also shown in FIG. 3).

SEQ ID NO. 4 is a polypeptide encoded by SEQ ID NO. 3, which can be usedaccording to the subject invention.

SEQ ID NO. 5 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe according to the subject invention.

SEQ ID NO. 6 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe or PCR primer according to the subject invention.

SEQ ID NO. 7 is an oxalyl-CoA decarboxylase 5'-primer, which can be usedaccording to the subject invention.

SEQ ID NO. 8 is an oxalyl-CoA decarboxylase 3'-primer, which can be usedaccording to the subject invention.

SEQ ID NO. 9 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe or primer according to the subject invention.

SEQ ID NO. 10 is a formyl-CoA transferase sequence, which can be used asa probe according to the subject invention.

SEQ ID NO. 11 is an oxalyl-CoA decarboxylase sequence, which can be usedas a PCR primer according to the subject invention.

SEQ ID NO. 12 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe according to the subject invention.

SEQ ID NO. 13 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe according to the subject invention.

SEQ ID NO. 14 is an oxalyl-CoA decarboxylase sequence, which can be usedas a probe according to the subject invention.

SEQ ID NO. 15 is an oxalyl-CoA decarboxylase sequence, which can be usedas a PCR primer according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides an accurate, sensitive assay for oxalatein biological samples such as urine and serum. Elevated levels ofoxalate are correlated with urinary tract stone formation, as well asother health problems. Early detection of high levels of oxalate makesit possible to prevent, delay or reduce adverse health consequencesthrough appropriate medication and through modulation of diet.

In the presently described diagnostic system, two enzymes are used tocatabolize oxalate to carbon dioxide and formate. Specifically, anyoxalate that may be present in a sample being assayed is converted intoformate and carbon dioxide (CO₂) through the combined action of theenzymes oxalyl-CoA decarboxylase and formyl-CoA transferase. The formatecan then be detected using a variety of techniques known in the art. Ina preferred embodiment, the production of formate is measuredcolorimetrically by linking the catabolism of formate with theproduction of a detectable color change (for example, the formation of acompound that absorbs a particular wavelength of light). The productionof formate is directly correlated with the amount of oxalate present inthe sample. Therefore, if a known amount of formate is produced usingthe subject enzyme system, then the amount of oxalate present in thesample can be easily quantitated.

In a preferred embodiment, the enzymes used in the subject invention areexpressed by genes from the bacterium Oxalobacter formigenes. The genesencoding both oxalyl-CoA decarboxylase (Lung et al., 1994) andformyl-CoA transferase enzymes have been cloned and expressed, thusproviding a readily-available source of reagent material. The subjectassay is capable of detecting oxalate levels in a range as low as0.00025-0.0005 mM (FIG. 1). This level of sensitivity makes the subjectassay capable of direct detection of oxalate in serum samples consistingof little as 10 μl volume. The described system can be easily automatedwith standard systems known in the art.

In a preferred embodiment of the subject assay, the enzymatic reactioncan be carried out in the wells of flat-bottomed 96-well microtiterplates and read in an automated plate reader. Suitable concentrations ofthe assay reagents oxalyl-CoA decarboxylase, oxalyl-CoA, β-NAD, formatedehydrogenase, and the sample to be assayed are added to the microtiterwells. The reaction is then brought to equilibrium (two minuteincubation at 37° C. in the plate reader) to permit degradation of anyresidual formate that may be present in the sample. The formyl-CoAtransferase enzyme is then added to the mixture to start the reaction,and the plate is read at 15 second intervals. Formate production isdetermined by measuring the reduction in NAD in the presence of formatedehydrogenase by detecting changes in absorbance of the sample at 340 nm(Baetz and Allison, 1989). The quantity of oxalate is determined bycomparison of the unknown samples with standards having a known amountof oxalate.

Further, the enzymatic reaction of the subject assay will not beinitiated until the formyl-CoA transferase, oxalyl-CoA decarboxylase,and oxalyl-CoA are all present within the reaction mixture. Therefore,initiation of the enzymatic reaction can be prevented by withholding oneof the above reagents from the reaction mix. Preferably, oxalyl-CoAdecarboxylase and oxalyl-CoA are added first, and the reaction isinitiated by the addition of formyl-CoA transferase to the mix. However,the order of addition of the three reagents is not material to thefunction of the assay, so long as one of the reagents is withheld untiljust prior to the desired initiation point of the assay.

The formyl-CoA transferase and oxalyl-CoA decarboxylase enzymes used inthe subject invention can be obtained and purified as a natural productof Oxalobacter formigenes (Baetz and Allison, 1989 and 1990).Alternatively, the enzymes can be obtained from host cells expressingthe recombinant polynucleotide molecules of the subject invention thatencode the enzymes. Other reagents used in the subject assay can beobtained from conventional sources, such as Sigma Chemical Company, St.Louis, Mo. Further, a person of ordinary skill in the art can readilydetermine the optimal concentrations of the reagents to use in the assaydescribed herein.

A further aspect of the subject invention concerns the cloning,sequencing and expression of the Oxalobacter formigenes gene whichencodes the formyl-CoA transferase used in the assay that is a subjectof the invention. The gene was cloned using degenerate oligonucleotideprobes (based on partial amino acid sequencing of tryptic peptides) toscreen an Oxalobacter genomic DNA library. The gene encodes apolypeptide having a molecular weight of approximately 40 kD. Thesubject invention further concerns the cloning, sequencing, andexpression of the gene which encodes oxalyl-CoA decarboxylase fromOxalobacter formigenes. The nucleotide sequence of the cDNA offormyl-CoA transferase and oxalyl-CoA decarboxylase are shown in FIGS. 2and 3, respectively (SEQ ID NOS. 1 and 3).

Because of the redundancy of the genetic code, a variety of differentpolynucleotide sequences can encode the formyl-CoA transferasepolypeptide disclosed herein. It is well within the skill of a persontrained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, polypeptide of the subjectinvention. These variant or alternative polynucleotide sequences arewithin the scope of the subject invention. As used herein, references to"essentially the same" sequence refers to sequences which encode aminoacid substitutions, deletions, additions, or insertions which do notmaterially alter the functional enzymatic activity of the encodedpolypeptide. Further, the subject invention contemplates thosepolynucleotide molecules having sequences which are sufficientlyhomologous with the DNA sequences shown in FIGS. 2 and 3 (SEQ ID NOS. 1and 3) so as to permit hybridization with those sequences under standardhigh-stringency conditions. Such hybridization conditions areconventional in the art (see, e.g., Maniatis et al., 1989).

As a person skilled in the art would appreciate, certain amino acidsubstitutions within the amino acid sequence of the polypeptide can bemade without altering the functional activity of the enzyme. Forexample, amino acids may be placed in the following classes: non-polar,uncharged polar, basic, and acidic. Conservative substitutions, wherebyan amino acid of one class is replaced with another amino acid of thesame class, fall within the scope of the subject invention so long asthe substitution does not materially alter the enzymatic reactivity ofthe polypeptide. Non-conservative substitutions are also contemplated aslong as the substitution does not significantly alter the functionalactivity of the encoded polypeptide.

The polynucleotides of the subject invention can be used to express therecombinant formyl-CoA transferase enzyme. They can also be used as aprobe to detect related enzymes. The polynucleotides can also be used asDNA sizing standards.

The polypeptides encoded by the polynucleotides can be used to raise animmunogenic response to the formyl-CoA transferase enzyme. They can alsobe used as molecular weight standards, or as inert protein in an assay.The polypeptides can also be used to detect the presence of antibodiesimmunoreactive with the enzyme.

The polynucleotide sequences of the subject invention may be composed ofeither RNA or DNA. More preferably, the polynucleotide sequences arecomposed of DNA. The subject invention also encompasses thosepolynucleotides that are complementary in sequence to the polynucleotidesequences disclosed herein.

Another aspect of the subject invention pertains to kits for carryingout the enzyme assay for oxalate. In one embodiment, the kit comprises,in packaged combination and in relative quantities to optimize thesensitivity of the described assay method, (a) the oxalyl-CoAdecarboxylase, oxalyl-CoA, p-NAD, and formate dehydrogenase; and (b)formyl-CoA transferase. The kit may optionally include other reagents orsolutions, such as buffering and stabilization agents, along with anyother reagents that may be required for a particular signal generationsystem. Other reagents such as positive and negative controls can beincluded in the kit to provide for convenience and standardization ofthe assay method.

The subject invention further concerns a method for detecting thepresence of Oxalobacter formigenes organisms in a sample. Specificpolynucleotide probes can be prepared based on the nucleotide sequenceof either the oxalyl-CoA decarboxylase or the formyl-CoA transferasegene sequence of Oxalobacter formigenes. The polynucleotide probes ofthe subject invention can be used to identify Oxalobacter formigenes ina sample, and to classify the strain of Oxalobacter formigenes detected.The polynucleotide probes of the subject invention can be used accordingto standard procedures and conditions to specifically and selectivelydetect polynucleotide sequences that have sufficient homology tohybridize with the probe. DNA can be isolated from bacterialmicroorganisms in a biological specimen (e.g., biopsy, fecal matter,tissue scrapings, etc.) using standard techniques known in the art andthe isolated DNA screened for hybridization with Oxalobacter oxalyl-CoAdecarboxylase-specific and/or formyl-CoA transferase-specificpolynucleotide probes. Various degrees of stringency can be employedduring the hybridization, depending on the amount of probe used forhybridization, the level of complementarity (i.e., homology) between theprobe and target DNA fragment to be detected. The degree of stringencycan be controlled by temperature, ionic strength, pH, and the presenceof denaturing agents such as formamide during hybridization and washing.Hybridization methods and conditions are known in the art and aregenerally described in Nucleic Acid Hybridization: A Practical Approach(Hames, B. D., S. J. Higgins, eds.), IRL Press (1985).

The polynucleotide probes of the subject invention include, for example,the oxalyl-CoA decarboxylase probe A (SEQ ID NO. 5), probe AP15 (SEQ IDNO. 6), and probe AP34 (SEQ ID NO. 9), probe AP286 (SEQ ID NO. 12),probe AP307 (SEQ ID NO. 13), and probe HS-2 (SEQ ID NO. 14),specifically exemplified herein. Probes for formyl-CoA transferaseinclude, for example, probe AP273 (SEQ ID NO. 10) specificallyexemplified herein. The nucleotide sequences of the exemplified probesare shown below:

                         (SEQ ID NO. 5)                                           Probe A  5'-GAGCGATACCGATTGGA-3'                                                                   (SEQ ID NO. 6)                                           Probe AP15                                                                             5'-GCACAATGCGACGACGA-3'                                                                   (SEQ ID NO. 9)                                           Probe AP34                                                                             5'-ATACTCGGAATTGACGT-3'                                                                   (SEQ ID NO. 10)                                          Probe AP273                                                                            5'-TTCATGTCCAGTTCAATCGAACG-3'                                                             (SEQ ID NO. 12)                                          Probe AP286                                                                            5'-GACAATGTAGAGTTGACTGATGGCTTTCATG-3'                                                     (SEQ ID NO. 13)                                          Probe AP307                                                                            5'-CAGGATGGTCAGAAGTTC-3'                                                                  (SEQ ID NO. 14)                                          Probe HS-2                                                                             5'-CCGGTTACATCGAAGGA-3'                                          

The polynucleotide probes contemplated in the subject invention alsoinclude any polynucleotide molecule comprising a nucleotide sequencecapable of specifically hybridizing with oxalyl-CoA decarboxylase orformyl-CoA transferase polynucleotide sequence of the present invention.As used herein, reference to "substantial homology" or "substantiallycomplementary" refers not only to polynucleotide probes of the subjectinvention having 100% homology with the nucleotide sequence of thetarget polynucleotide, or fragments thereof, but also to those sequenceswith sufficient homology to hybridize with the target polynucleotide.Preferably, the degree of homology will be 100%; however, the degree ofhomology required for detectable hybridization will vary in accordancewith the level of stringency employed in the hybridization and washes.Thus, probes having less than 100% homology to the oxalyl-CoAdecarboxylase or formyl-CoA transferase polynucleotide sequences can beused in the subject method under appropriate conditions of stringency.In a preferred embodiment, high stringency conditions are used. Inaddition, analogs of nucleosides may be substituted for naturallyoccurring nucleosides within the polynucleotide probes. Such probeshaving less than 100% homology or containing nucleoside analogs arewithin the scope of the subject invention. The skilled artisan, havingthe benefit of the disclosure contained herein, can readily prepareprobes encompassed by the subject invention.

In addition, the subject invention also concerns polynucleotide primersthat can be used for polymerase chain reaction (PCR) amplification ofOxalobacter formigenes nucleotide sequences. PCR amplification methodsare well known in the art and are described in U.S. Pat. Nos. 4,683,195;4,683,202; and 4,800,159. In a preferred embodiment, the polynucleotideprimers are based on the oxalyl-CoA decarboxylase or formyl-CoAtransferase gene sequence and can be used to amplify the full length ora portion of the target gene. The amplified Oxalobacter sequences can bedetected using the probes of the subject invention according to standardprocedures known in the art.

The polynucleotide primers of the subject invention include, forexample, oxalyl-CoA decarboxylase PCR primer 1 (SEQ ID NO. 7), PCRprimer 2 (SEQ ID NO. 8), PCR primer AP15 (SEQ ID NO. 6), and PCR primerAP22 (SEQ ID NO. 11), PCR primer AP34 (SEQ IS NO. 9), and PCR primerAP21 (SEQ ID NO. 15), specifically exemplified herein. The nucleotidesequences of the exemplified PCR primers are shown below:

                         (SEQ ID NO. 7)                                           PCR primer 1                                                                              5'-CAGGTTATGCAGCTTCT-3'                                                                (SEQ ID NO. 8)                                           PCR primer 2                                                                              5'-GGATGGTTGTCAGGCAG-3'                                                                (SEQ ID NO. 6)                                           PCR primer AP15                                                                           5'-GCACAATGCGACGACGA-3'                                                                (SEQ ID NO. 11)                                          PCR primer AP22                                                                           5'-GTAGTTCATCATTCCGG-3'                                                                (SEQ ID NO. 9)                                           PCR primer AP34                                                                           5'-ATACTCGGAATTGACGT-3'                                                                (SEQ ID NO. 15)                                          PCR primer AP21                                                                           5'-TCCAATCGGTATCGCTC-3'                                       

The primer pair AP34 (SEQ ID NO. 9) and AP21 (SEQ ID NO. 15) (derivedfrom oxc sequences between bp -59 to -41 and by 451 to 435,respectively), consistently amplifies a 500 bp segment of oxc from allO. formigenes strains and isolates tested. PCR application of wholefecal DNA with this genus-specific primer pair, in conjunction withSouthern Blotting using genus and group specific probes, now provides arapid diagnostic tool to detect and speciate O. formigenes.Time-consuming steps, e.g., agarose-gel electrophoresis and Souther blothybridizations, can be substituted with newer technologies such asmicrotiter-plate based colorimetric or fluorogenic assays (Jordan etal., 1996).

Polynucleotide primers contemplated by the subject invention alsoinclude any polynucleotide molecule comprising a nucleotide sequencecapable of specifically priming amplification of oxalyl-CoAdecarboxylase or formyl-CoA transferase polynucleotide sequencesdisclosed herein. As used herein, reference to "substantial homology" or"substantially complementary" refers not only to polynucleotide primersof the subject invention having 100% homology with the nucleotidesequence of the target polynucleotide, or fragments thereof, but also tothose sequences with sufficient homology to hybridize with and prime theamplification of a target polynucleotide. Preferably, the degree ofhomology will be equal to or about 100%. The skilled artisan, having thebenefit of the disclosure contained herein, can readily prepare otherprimers of varying nucleotide length and sequence that can be used toamplify all or portions of the oxalyl-CoA decarboxylase and/or theformyl-CoA transferase gene.

The polynucleotide probes and primers of the subject invention can bechemically synthesized or prepared through recombinant means usingstandard methods and equipment. The polynucleotide probes and primerscan be in either single- or double-stranded form. If the probe or primeris double-stranded, then single-stranded forms can be prepared from thedouble-stranded form. The polynucleotide probes and primers may becomprised of natural nucleotide bases or known analogs of the naturalnucleotide bases. The probes and primers of the subject invention mayalso comprise nucleotides that have been modified to bind labelingmoieties for detecting the probe or primer or amplified gene fragment.

The polynucleotide molecules of the subject invention can be labeledusing methods that are known in the art. The polynucleotides may beradioactively labeled with an isotope such as ³ H, ³⁵ S, ¹⁴ C, or ³² PThe polynucleotides can also be labeled with fluorophores,chemiluminescent compounds, or enzymes. For example, a polynucleotidemolecule could be conjugated with fluorescein or rhodamine, or luciferinor luminol. Similarly, the polynucleotide molecule can be conjugatedwith an enzyme such as horseradish peroxidase or alkaline phosphatase.Polynucleotide molecules can also be detected by indirect means. Forexample, the polynucleotide may be conjugated with ligands, haptens, orantigenic determinants. The conjugated polynucleotide is then contactedwith the ligand receptor, with an anti-ligand molecule that binds to theligands, or with an antibody that binds to the hapten/antigenicdeterminant, respectively. For example, the polynucleotide can belabeled with digoxygenin and detected with labeled anti-digoxygeninantibodies. The ligand receptor, anti-ligand molecule, or antibody maybe directly labeled with a detectable signal system, such as afluorophore, chemiluminescent molecule, radioisotope, or enzyme. Methodsfor preparing and detecting labeled moieties are known in the art.

In one embodiment of the present detection method, samples to be testedfor the presence of Oxalobacter formigenes are obtained from a person oranimal, and DNA is isolated from the specimen using standard techniquesknown in the art. For example, cells can be lysed in an alkali solution,the nucleic acid extracted in phenol:chloroform, and then precipitatedwith ethanol. The DNA is then fragmented into various sizes usingrestriction endonuclease enzymes or other means known in the art. TheDNA fragments are then electrophoretically separated by size on anagarose gel. In an alternative embodiment, the DNA fragments aresubjected to PCR amplification using PCR primers of the presentinvention prior to gel electrophoresis in order to specifically amplifyportions of the formyl-CoA transferase and oxalyl-CoA decarboxylasegenes.

After the DNA fragments are separated on the gel, the size-fractionatedDNA fragments are transferred to a membrane matrix, such asnitrocellulose, nylon, or polyvinylidene difluoride (PVDF), by Southernblotting. The DNA immobilized on the membrane matrix is single-stranded.Polynucleotide probes of the subject invention are then contacted withthe membrane and allowed to hybridize with the DNA immobilized on themembrane. A probe of the present invention can be labeled with adetectable signal, such as a radioisotope, or the probe can be labeledwith a hapten or antigen such as digoxigenin. The hybridization can beperformed under conditions known in the art. After hybridization of theprobe with the DNA fragments on the membrane, the membrane is washed toremove non-hybridized probe. Standard wash conditions are known in theart, and the stringency and number of washes employed can vary.

The membrane is then tested or observed for the presence of hybridizedprobe. For example, if the hybridized probe was labeled with a hapten orantigen, then it can be detected using an antibody that binds to theconjugated hapten or antigen on the probe. The antibody can be directlylabeled with a detectable fluorophore, chemiluminescent molecule,radioisotope, enzyme, or other signal generating system known in theart. Alternatively, the antibody can be detected using a secondaryreagent that binds to the antibody, such as anti-immunoglobulin, proteinA, protein G, and other antibody binding compositions known in the art.The secondary reagent can be labeled with a detectable fluorophore,chemiluminescent molecule, radioisotope, or enzyme. The presence of adetectable hybridization signal on the membrane indicates the presenceof Oxalobacter formigenes in a test sample.

The subject invention also concerns a kit for the detection ofOxalobacter formigenes in a sample. A kit contemplated by the subjectinvention may include in one or more containers: polynucleotide probes,positive and negative control reagents, and reagents for detecting theprobes. The kit may also include polynucleotide primers for performingPCR amplification of specific Oxalobacter formigenes genes. In apreferred embodiment, the polynucleotide probes and primers are specificfor the oxalyl-CoA decarboxylase and formyl-CoA transferase genes of O.formigenes.

The subject invention also concerns a dipstick device comprising theenzymes of the subject invention and dyes and/or substrates immobilizedon a carrier matrix. Any dye or substrate that yields a detectableproduct upon exposure to the reaction products that are produced by theenzymatic reaction of oxalate with oxalyl-CoA decarboxylase andformyl-CoA transferase as described herein is contemplated for use withthe subject dipstick device. The carrier matrix of the assay device canbe composed of any substance capable of being impregnated with theenzyme and dye components of the subject invention, as long as thematrix is substantially inert with respect to the analyte being assayedfor. For example, the carrier matrix may be composed of paper,nitrocellulose, PVDF, or plastic materials and the like.

Incorporation of the enzymes, dye and other components on the carriermatrix can be accomplished by any method such as dipping, spreading orspraying. A preferred method is impregnation of the carrier matrixmaterial by dipping in a reagent solution and drying to remove solvent.Drying can be accomplished by any means which will not deleteriouslyaffect the reagents incorporated, and typically is by means of an airdrying oven.

The dipstick device of the subject invention is dipped in or contactedwith a sample to be tested for the presence or amount of oxalate.Positive and negative controls can be used in conjunction with thedipstick device. An appropriate amount of time is allowed to pass andthen the dipstick is assessed for a positive reaction by visualinspection. If oxalate is present in the sample then a detectablesignal, usually in the form of a color, can be observed on the dipstick.Typically, the intensity of the color developed in a fixed time periodis proportional to the concentration of oxalate present in the sample.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Determination of Level of Sensitivity of Enzyme Assay System

Samples containing oxalate at concentrations ranging from 0.004 mM to0.00025 mM were prepared in 10 μl volumes. The samples were then assayedusing the enzyme system of the subject invention in 96-well microtiterplates. Reagents were then added at the following concentrations: KH₂PO₄ (pH 6.7), 50 mM; MgCl₂, 5 mM; thiamine PPi (TPP), 2 mM; oxalyl-CoA,0.375 mM; β-NAD, 1.0 mM; formate dehydrogenase, 0.25 IU; and oxalyl-CoAdecarboxylase, 0.03 U. The reaction mixture was then incubated at 37° C.for 2 minutes in order to permit the degradation of any residual formatethat may be present in the sample mixture. The reaction was theninitiated by the addition of formyl-CoA transferase to the samplemixture. Changes in A₃₄₀ were measured every 15 seconds at 37° C. (FIG.1). Appropriate positive and negative controls were run simultaneouslywith the assay.

EXAMPLE 2 Detection of Oxalobacter formigenes in a Sample

Strains of Oxalobacter formigenes used in the following methods arelisted in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Description of the Oxalobacter formigenes strains                             Group Classification of Source of                                             O. formigenes strains.sup.a                                                                 Strain    Isolate                                               ______________________________________                                        Group I       OxB       Sheep rumen                                                         OxWR      Wild rat cecum                                                      SOx-4     Freshwater lake sediment                                            SOx-6     Freshwater lake sediment                                            POxC      Pig cecum                                                           HC-1      Human feces                                           Group II      BA-1      Human feces                                                         OxK       Human feces                                                         HOxBLS    Human feces                                                         HOxRW     Human feces                                                         OxCR      Lab rat cecum                                                       OxGP      Guinea pig cecum                                      ______________________________________                                         .sup.a From Jensen and Allison (1994).                                   

All Oxalobacter formigenes strains were grown in medium B containing 30mM oxalate, as described in Allison et al. (1985). Human fecal samples(approximately 60 mg) were inoculated anaerobically into vialscontaining 9 ml of media B, then sequentially transferred through 10⁻⁸dilutions. Cultures were incubated at 37° C. for 10 days andbiochemically tested for the catabolic consumption of oxalate by CaCl₂precipitation (50 μl media, 100 μl 1% CaCl₂, and 2.7 ml dH₂ O) andspectrophotometric analyses (600 nm).

Cultures (10-15 ml) of O. formigenes were centrifuged at 10,000×g, thebacterial pellet was resuspended in 567 μl TE buffer (10 mM Tris-Cl, pH7.5 plus 1 mM EDTA, pH 8.0), 30 μl 10% sodium dodecyl sulfate (SDS) and3 μl of proteinase K (20 mg/ml), and the mixture incubated 5 hr at 37°C. to ensure bacterial cell lysis. Nucleic acids were extracted from thelysates using phenol/chloroform/isoamylalcohol (25:24:1). ChromosomalDNA was precipitated from the aqueous phase by adding 1/2 volume of 7.5M ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered bycentrifugation(12,000×g), washed once with 70% ethanol, and the pelletresuspended in 15-20 μl H₂ O. Bacterial DNA was also isolated directlyfrom fresh human stool samples following lysis with chaotropic salt andguanidine thiocyanate, then binding to glass matrix (GlasPac, NationalScientific Supply, San Rafael, Calif.) (Stacy-Phips et al., 1995).

Bacterial DNA was digested with the restriction endonuclease Hind III(Life Technologies, Inc., Gaithersburg, Md.). The restriction-enzymegenerated fragments were size separated by gel electrophoresis through0.5% agarose, stained with ethidium bromide (EtBr), illuminated with UVlight, and photographed to document proper digestion. Digested DNA wasthen transferred from the agarose gels to positively-charged nylonmembranes (Boehringer-Mannheim GmBH, Indianapolis, Ind.) by positivepressure blotting and UV cross-linking (Stratagene, LaJolla, Calif.).Hybridizations were carried out using internal sequence oligonucleotideprobes. Oligonucleotides were synthesized in the University of FloridaICBR Oligonucleotide Synthesis Laboratory (Gainesville, Fla.) and havethe sequences:

                         (SEQ ID NO. 6)                                           AP15 5'-GCACAATGCGACGACGA-3'                                                                       (SEQ ID NO. 11)                                          AP22 5'-GTAGTTCATCATTCCGG-3'                                                                       (SEQ ID NO. 9)                                           AP34 5'ATACTCGGAATTGACGT-3'                                                                        (SEQ ID NO. 10)                                          AP273                                                                              5'-TTCATGTTCCAGTTCAATCGAACG-3'.                                      

Each oligonucleotide was end-labeled with digoxigenin in a reactionusing terminal transferase. The digoxigenin-labeled oligonucleotideprobes were hybridized to the immobilized DNA fragments andhybridization detected calorimetrically by enzyme-linked immunoassay(ELISA) using an anti-digoxigenin alkaline phosphatase conjugateaccording to the manufacturer's protocol provided with the GENIUS IIIdetection system (Boehringer-Mannheim).

All PCRs were performed according to protocols described in Anderson etal. (1993). Briefly, 50 μl reactions contained 1.5 mM MgCl₂, 200 μMdNTP, 1.25 U Taq polymerase (GIBCO-BRL, Bethesda, Md.), 1 μg templateDNA and 1 μM each of a 5' and 3' primer. A preferred reaction profileproved to be 94° C. for 5 min, then 45 cycles of 94° C. for 1 min ofdenaturation, 55° C. for 2 min of annealing and 72° C. for 3 min ofprimer extension. PCR products were size separated by gelelectrophoresis in 1.2% agarose containing EtBr and photographed in UVlight. PCR primer AP15 (SEQ ID NO. 6) and primer AP22 (SEQ ID NO. 11)were used as primers.

Previous studies by Lung et al. (1994) showed that genomic DNA of O.formigenes, strain OxB, could be digested with the restriction enzymeHind III and that a limited number of enzyme cleavage sites existed nearor within the oxalyl-CoA decarboxylase (oxc) gene. A RFLP analysis ofHind III digested OxB genomic DNA using either probe AP15 (SEQ ID NO.6), a probe homologous to an internal sequence of the oxc gene, probeAP34 (SEQ ID NO. 9), a probe homologous to a 5'-end sequence of the oxcgene but separated from the probe AP15 (SEQ ID NO. 6) sequence by a HindIII site, or probe AP273 (SEQ ID NO. 10), a probe homologous to aninternal sequence of the formyl-CoA transferase (frc) gene, is shown inFIG. 4. Using probe AP15 (SEQ ID NO. 6), a fragment of approximately 7kb containing a portion of the oxc gene was detected, while fragments ofapproximately 3 kb were detected using either probe AP34 (SEQ ID NO. 9)or probe AP273 (SEQ ID NO. 10). The 3 kb fragment identified by probeAP34 (SEQ ID NO. 9) is distinct from the 3 kb fragment detected by probeAP273 (SEQ ID NO. 10).

As shown in FIG. 5, the oxalyl-CoA decarboxylase and formyl-CoAtransferase genes were consistently detected in samples containing aslittle as 0.06 to 0.20 μg of O. formigenes, strain OxB, DNA orapproximately 0.20 to 0.40 μg of O. formigenes DNA from other group Istrains, such as HC-1. The 23-bp probe AP273 (SEQ ID NO. 10) can detectthe frc gene in DNA samples containing only one-fourth the amount of DNArequired for the 13 bp probe AP15 (SEQ ID NO. 6) to detect the oxc gene(FIG. 5, upper panel). These probes are highly specific for O.formigenes since they fail to bind to other bacterial DNA, includingEscherichia coli, Alcaligenes oxalaticus, and fecal bacteroides.

Protein, lipid and genetic studies of several isolates of O. formigeneshave provided the basis for dividing this genus into two majorsubgroupings (Jensen et al., 1994). When RFLP analyses were performed ongenomic DNA isolated from various Oxalobacter formigenes strains, probesAP15 (SEQ ID NO. 6) and AP273 (SEQ ID NO. 10) were able to distinguishgroup I strains from group II strains on the Southern blothybridizations (FIG. 6). All strains of O. formigenes belonging to groupI (to which OxB is assigned) hybridized with both probe AP15 (SEQ ID NO.6) and probe AP273 (SEQ ID NO. 10). Due to a characteristic slow growthof strain HC-1, only faint bands appeared in this experiment. Incontrast, none of the O. formigenes strains assigned to group IIhybridized with probe AP273 (SEQ ID NO. 10) and only BA-1 hybridizedwith probe AP15 (SEQ ID NO. 6). These data indicate a highly conservedhomology of oxc and frc within group I strains and a less conservedhomology within group II strains.

To increase the sensitivity of detecting O. formigenes, PCR was used toamplify that portion of oxc which by RFLP appeared to differentiate thegroup I and group II strains. Using primer AP15 (SEQ ID NO. 6) andprimer AP22 (SEQ ID NO. 11) as PCR primers to amplify a DNA segment inthe carboxy-terminal region of oxc, strains assigned to group I (i.e.,OxB, HC-1, OxWR, POxC, SOx-4 and SOx-6) exhibited a common band at 452bp (FIG. 7). In contrast, the other six strains, all belonging to groupII, showed variable amplification patterns, but all showed a dominantPCR band of approximately 630 bp, with a weaker 452 bp band. Sequenceanalysis of this 630 bp band from strain OxK has revealed the presenceof the 452 bp sequence present in the 630 bp PCR product. Close analysisof the group II strains suggest that their PCR amplification profilesare highly reproducible, suggesting group II strains may fall into three(sub)groupings: HOxBLS and HOxRW (subgroup 1), OxCR and OxGP (subgroup2), and BA-1 and OxK (subgroup 3).

The use of PCR-based detection of the oxc gene to identify O. formigenesin clinical specimens was examined by comparing PCR and biochemicalmethods of detection. Specimen 1, known to be positive for O.formigenes, gave ambiguous results in biochemical testing for oxalatedepletion, but exhibited the presence of the 450 bp PCR productindicative of an O. formigenes group I strain. Specimen 2, known to benegative for O. formigenes, proved negative using both PCR-based andbiochemical testing. Specimen 3, known to be positive for O. formigenes,showed depletion of oxalate in all dilutions and revealed a PCR patternsuggestive of an O. formigenes group II strain. PCR amplification wasnot observed in the original culture or the first dilution due to thepresence of inhibitors of PCR e.g., bile salts, bilirubin, etc.) whichcopurify with DNA.

To circumvent the inhibition of the PCR by factors co-purifying with thebacterial DNA, DNA isolation was performed by lysing fresh stool sampleswith guanidine thiocyanate followed by adsorption to and elution fromglass matrices. Using this method, PCR-based detection of O. formigenescan be performed using fecal DNA diluted only 1:25 to 1:50 to eliminatePCR inhibitors. Sensitivity experiments using different stool samplesspiked with strains OxB or OxK in the range of 10¹ to 10⁷ cfu per 0.1 gof sample showed that as few as 10² to 10³ cfu of O. formigenes per 0.1g sample could be detected (FIG. 8). Again, PCR-based analyses of DNAisolated directly from a stool sample known to be positive for O.formigenes by culture methods, showed amplification patterns indicativeof a group II strain (FIG. 8, lanes F & G).

EXAMPLE 3 Detection and Classification of Oxalobacter formigenes

Bacterial Strains

O. formigenes strains used included OxB (isolated from sheep rumen) andHC1, OxK, BA1, HOxBLS, HOxRW, HOxRA, HOxCC13, and HOxHM18 (isolated fromhuman feces). In addition, several new purified cultures, includingHOxUK5, HOxUK88, HOxUK90, and HOxHS (grown from human feces), were alsoused. All strains and isolates were grown in media B containing 30 mMpotassium oxalate, as described elsewhere (Allison et al., 1985), andmaintained under strict anaerobic conditions until used.

Preparation of Genomic DNA from O. formigenes Cultures

Fifteen ml cultures of O. formigenes were centrifuged at 10,000×g, thebacterial pellet resuspended in 567 μl of TE buffer (10 mM Tris-HC1, pH7.5, plus 1 mM EDTA, pH 8.0), 30 μl of 10% sodium dodecyl sulfate plus 3μl of proteinase K (20 mg/ml), and this mixture incubated for 5 hours at37° C. to ensure bacterial cell lysis. Nucleic acids were extracted fromthe lysates with phenol:chloroform:isoamylalcohol (25:24: 1).Chromosomal DNA was precipitated by adding 1/2 volume of 7.5 M ammoniumacetate and 2 volumes of 100% ethanol. DNA was recovered bycentrifugation(12,000×g) and washed once in 70% ethanol. The final DNAprecipitation was resuspended in 20 μl H₂ O.

Sequence Analysis of the oxc Genes

The primer pair,

5'-ATACTCGGAATTGACGT-3' (a 5'-primer designated AP34) (SEQ ID NO. 9) and

5'-TCCAATCGGTATCGCTC-3' (a 3'-primer designated AP21) (SEQ ID NO. 15)

homologous to sequences within the 5'-end of the oxc gene present instrain OxB (Lung et al., 1994), was used to amplify a 500 bp DNAfragment from genomic DNA isolated from each of twelve human O.formigenes strains. Amplifications were performed in 50 μl PCR reactionscontaining 1.5 mM MgCl₂, 200 μM deoxynucleoside triphosphate, 1.25 U ofTaq polymerase (Gibco-BRL, Bethesda, Md.), 1 μg of genomic DNA and 1 μMeach of 5'- and 3'-primer. PCR were carried out for 35 cycles andincluded an initial 5 minute denaturation step at 94° C., 1 minuteannealing (with a temperature stepdown from 60° C. to 55° C.), 1 minuteextension at 72° C. and a final 8 minute extension at 72° C. The PCRproducts were size fractionated by electrophoreses through 1.2% agarosegels containing ethidium bromide for visualization of the bands in UVlight. Each 500 bp PCR product was cloned into the TA cloning system,pCR-2.1 (Invitrogen, Inc., San Diego, Calif.). Competent DH5E. colibacteria were transfected with the recombinant plasmid and transformedbacteria selected on LB agar plates containing 10 μl/ml of ampicillinand 20 mg/ml of X-Gal. DNA from appropriate clones was isolated, checkedfor the presence of an insert of correct size by digestion with therestriction enzyme, Eco RI. Inserts of recombinant plasmids weresequenced using M13-forward and M13-reverse primers.

Clinical Samples

Fecal samples of 100 generally healthy children of either sex ranging inage from 0 to 12 years were examined for the presence of O. formigenes.All fecal samples were collected in Dzerzhinsk, a city in the Donetskregion of the Ukraine. This particular population was selected due tothe fact that these children have had limited use of antibiotics, thatmight influence bacterial colonization of the intestinal tract, intreatment of childhood diseases. Approximately 25 mg sample of freshstool (within 3-4 hours of collection), was inoculated into vialscontaining 10 ml of anaerobically sealed media B supplemented to 30 mMwith potassium-oxalate. The vials were analyzed at the University ofFlorida, Gainesville, Fla. After incubation at 37° C. for one week, theloss of oxalate from each fecal culture was determined using acalcium-chloride precipitation method in which 50 μl culture media ismixed with 100 μl 0.1% CaCl₂ plus 3.0 ml dH₂ O and the absorbance ofeach mixture determined spectrophotometrically (600nm). The calciumprecipitation test for loss of oxalate has been repeatedly verified asreliable by other methods (e.g., gas chromatography and butyl esters)for detection of oxalate. Typically, cultures not showing catabolism ofoxalate generally have O.D. readings of about 0. 1, whereas cultureswith oxalate degradation have O.D. readings less than about 0.02.

PCR-based Detection and Identification of O. formigenes

DNA was isolated from individual fecal cultures by the method of Phippset al. (Stacy-Phipps et al., 1995) using guanidine thiocyanate as achaotropic agent and glass-matrix for DNA binding. One 1 μl of each DNAsample was used as template in a 50 μl PCR reaction as described above.The amplified PCR products were size separated by electrophoresisthrough 1.2% agarose gels containing ethidium bromide and visualizedwith UV light. Each reaction was controlled using a reaction containingall the components of the PCR with the exception of template DNA.

Southern Blot Analysis

Southern blots were carried out as previously detailed in Example 2.Briefly, the size separated PCR products were transferred to positivelycharged nylon membranes (Boehringer Mannheim GmBH, Indianapolis, Ind.)by positive pressure blotting and UV-crosslinking. The oxc derived genusspecific (AP286), group I specific (HS-2) and group II specific (AP307)oligonucleotides were synthesized in the University of Florida ICBR DNASynthesis Laboratory (University of Florida, Gainesville, Fla.) andend-labeled with digoxigenin in a reaction using terminal transferase.The digoxigenin labeled oligonucleotides were hybridized to theimmobilized PCR products under conditions of high stringency (5X SSC and68° C.). Hybridization was detected colorimetrically by enzyme-linkedimmunosorbent assay (ELISA) with an anti-digoxigenin alkalinephosphatase conjugate according to the manufacturer's protocol providedwith the GENIUS III kit (Boehringer Mannheim GmBH).

Generation of Genus-specific and Group-specific Probes

Preliminary studies looking at the efficacy of various oligonucleotidepairs to amplify portions of the oxc gene present in various O.formigenes strains revealed that the PCR primer pair AP34(5'-primer)/Ap21 (3'-primer) amplified a 500 bp DNA fragment in bothgroup I and group II strains. To determine the degree of sequencehomology within the 5'-end of the oxc gene between various strains of O.formigenes, genomic DNA was prepared from 5 group I and 7 group IIstrains isolated from human fecal samples for use as template in PCRwith AP34 and AP21. Each PCR amplified an expected 500 bp product thatwas subsequently cloned into the pCR-2.1 vector system and sequenced. Acomparison of the 5'-end sequences of the oxc gene from these 12 humanisolates with the OxB gene is shown in part in FIG. 9. The 5'-end of theoxc gene appears to be relatively conserved for a bacterial gene, withmost bp changes occurring in the wobble base such that the codontranslation is not altered. Nevertheless, there were enough sequencedifferences to demarcate group I strains from group II strains, thuspermitting selection of regions that are conserved within strains of aspecific group, but differ significantly from strains of the othergroup. Based on these conserved regions, genus-specific oligonucleotideprobes (for example, probe AP286, homologous to the region between bp 13and 43 of the open-reading frame), as well as group I-specific (forexample, probe HS2, homologous to the region between bp 197 and 214 ofthe open-reading frame) and group II-specific (for example, probe AP307,homologous to the region between bp 133 and 150 of the open-readingframe) probes were prepared.

Specificity of the Genus-specific and Group-specific OligonucleotideProbes

The specificity of probes AP286, AP307, and HS2 in detecting andclassifying O. formigenes was examined using genomic DNA prepared fromnumber of known strains and isolates. PCR amplifications with thegenus-specific primer pair AP34 and AP21 resulted in the 500 bpamplification product in all cultures tested (FIG. 10A, top panel). OnSouthern blotting, this 500 bp fragment hybridized with a genus-specificprobe, AP286 (FIG. 10B, bottom panel).

In a separate experiment, the amplified 500 bp PCR product washybridized with either the group I-specific probe, HS2, (FIG. 11 B,middle panel) or the group II-specific probe, AP307, (FIG. 11C, bottompanel). Results clearly show a group specificity in the binding of thesegroup-specific probes and their ability to identify subgroups of O.formigenes.

Application of a PCR-based Detection System for O. formigenes

In a double-blinded study, 100 fecal samples were collected fromchildren ranging in age from newborn to 12 years and tested for thepresence of O. formigenes using both an oxalate degradation system andour PCR-based assay system. The aim of this study was to determine theage at which children become naturally colonized with this intestinalanaerobic bacterium. Of the 100 fecal samples examined, 72 samplestested positive for O. formigenes by PCR, 59 of which also exhibitedoxalate degradation in an oxalate degradation assay. Interestingly, ofthe 72 positive samples, 68 were group II strains while only 4 weregroup I strains. All fecal cultures exhibiting degradation of oxalatetested positive for O. formigenes by PCR. Although there were 13cultures that failed to degrade oxalate that proved positive for O.formigenes by PCR, the majority of the samples that failed to degradeoxalate also failed to exhibit amplification of a product in thePCR-reaction. These data show that the PCR-based assay is probably moresensitive than the biochemical (calcium chloride precipitation) test,yet highly specific.

When the data were unblinded, a clear pattern for the naturalcolonization of children became evident. O. formigenes could not bedetected in infants less than 6-9 months of age. O. formigenes beganappearing in the intestinal tracts of children around 1 year of age, andby 3-4 years of age, all children showed signs of being colonized.Although the sample size is small, the number of children colonized withO. formigenes declined between 8-12 years of age, reaching thecolonization frequency of 70-80% estimated for adult populations (Doaneet al., 1989,Kleinschmidt et al., 1993, Allison et al., 1986,and Goldkinet al, 1985).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

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Jordan, J. A., M. B. Durso (1996) "Rapid speciation of the five mostmedically relevant candida species using PCR amplification and amicrotitre plate-based detection system," Mol Diagnosis 1:51-58.

Kleinschmidt K., A. Mahlmann, R. Hautmann (1993) "Anaerobicoxalate-degrading bacteria in the gut decrease faecal and urinaryoxalate concentrationsin stone formers," In R. Ryall, R. Bais, V. R.Marshall, A. M. Rofe, L. H. Smith, V. R. Walker Urolithiasis 2, PlenumPress, New York, pp. 439-441.

Lung, H., A. L. Baetz, A. B. Peck (1994) "Molecular Cloning, DNASequence and Gene Expression of the Oxalyl-CoA Decarboxylase Gene, oxc,from the Bacterium Oxalobacter formigenes," J Bacteriol.176(8):2468-2472.

Maniatis, T., E. F. Fritsch, J. Sambrook (1989) Molecular Cloning: ALaboratory Manual, 2d Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

Smith, R. L., F. E. Strohmaier, R. S. Oremland (1985) "Isolation ofanaerobic oxalate-degrading bacteria from fresh water lake sediments,"Arch Microbiol 141:8-13.

Stacy-Phips, S., J. J. Mecca, J. B. Weiss (1995) "Multiplex PCR assayand simple preparation method for stool specimens detect enterotoxigenicE. coli DNA during course of infection," J Clin.

Microbiol 33:1054-1059.

Yriberri, J., L. S. Posten (1980) "A semi-automatic enzymic method forestimating urinary oxalate," Clin. Chem. 26(7):881-884.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 15                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1577 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - AAGCTTGCTT CATTTTGAGA TGTTATGCGA AGTGTTAGCA ACCCAAGTTA GT - #ACCCTTCA         60                                                                          - GCCCTTTGGG CGAAGTTTTT CTTTCTTGGC AGTTCCTTTC GGGGAAACAG CA - #CAGAGAAT        120                                                                          - AAAAACCAAA AGTTGTACCA ACGACAAGGA AATGAGAAAT TATGACTAAA CC - #ATTAGATG        180                                                                          - GAATTAATGT GCTTGACTTT ACCCACGTCC AGGCAGGTCC TGCCTGTACA CA - #GATGATGG        240                                                                          - GTTTCTTGGG CGCAAACGTC ATCAAGATTG AAAGACGTGG TTCCGGAGAT AT - #GACTCGTG        300                                                                          - GATGGCTGCA GGACAAACCA AATGTTGATT CCCTGTATTT CACGATGTTC AA - #CTGTAACA        360                                                                          - AACGTTCGAT TGAACTGGAC ATGAAAACCC CGGAAGGCAA AGAGCTTCTG GA - #ACAGATGA        420                                                                          - TCAAGAAAGC CGACGTCATG GTCGAAAACT TCGGACCAGG CGCACTGGAC CG - #TATGGGCT        480                                                                          - TTACTTGGGA ATACATTCAG GAACTGAATC CACGCGTCAT TCTGGCTTCC GT - #TAAAGGCT        540                                                                          - ATGCAGAAGG CCACGCCAAC GAACACCTGA AAGTTTATGA AAACGTTGCA CA - #GTGTTCCG        600                                                                          - GCGGTGCTGC AGCTACCACC GGTTTCTGGG ATGGTCCTCC AACCGTTTCC GG - #CGCTGCTC        660                                                                          - TGGGTGACTC CAACTCCGGT ATGCACCTGA TGATCGGTAT TCTGGCCGCT CT - #GGAAATGC        720                                                                          - GTCACAAAAC CGGCCGTGGT CAGAAAGTTG CCGTCGCTAT GCAGGACGCT GT - #TCTGAATC        780                                                                          - TGGTTCGTAT CAAACTGCGT GACCAGCAAC GTCTGGAAAG AACCGGCATT CT - #GGCTGAAT        840                                                                          - ACCCACAGGC TCAGCCTAAC TTTGCCTTCG ACAGAGACGG TAACCCACTG TC - #CTTCGACA        900                                                                          - ACATCACTTC CGTTCCACGT GGTGGTAACG CAGGTGGCGG CGGCCAGCCA GG - #CTGGATGC        960                                                                          - TGAAATGTAA AGGTTGGGAA ACCGATGCGG ACTCCTACGT TTACTTCACC AT - #CGCTGCAA       1020                                                                          - ACATGTGGCC ACAGATCTGC GACATGATCG ACAAGCCAGA ATGGAAAGAC GA - #CCCAGCCT       1080                                                                          - ACAACACATT CGAAGGTCGT GTTGACAAGC TGATGGACAT CTTCTCCTTC AT - #CGAAACCA       1140                                                                          - AGTTCGCTGA CAAGGACAAA TTCGAAGTTA CCGAATGGGC TGCCCAGTAC GG - #CATTCCTT       1200                                                                          - GCGGTCCGGT CATGTCCATG AAAGAACTGG CTCACGATCC TTCCCTGCAG AA - #AGTTGGTA       1260                                                                          - CCGTCGTTGA AGTTGTCGAC GAAATTCGTG GTAACCACCT GACCGTTGGC GC - #ACCGTTCA       1320                                                                          - AATTCTCCGG ATTCCAGCCG GAAATTACCC GTGCTCCGCT GTTGGGCGAA CA - #TACCGACG       1380                                                                          - AAGTTCTGAA AGAACTGGGT CTTGACGATG CCAAGATCAA GGAACTGCAT GC - #AAAACAGG       1440                                                                          - TAGTTTGATC CGTCAGACTT TCTGGGCAAA ACGGCACTCT CCGGAGTGCC GT - #TTTTTGTC       1500                                                                          - ACACGAAACC TAATCAAACA AGCACGTGCA ATGATTCCAC ATCATTGCGG CC - #ACATTCAT       1560                                                                          # 1577             G                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 428 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Thr Lys Pro Leu Asp Gly Ile Asn Val Le - #u Asp Phe Thr His Val         #                15                                                           - Gln Ala Gly Pro Ala Cys Thr Gln Met Met Gl - #y Phe Leu Gly Ala Asn         #            30                                                               - Val Ile Lys Ile Glu Arg Arg Gly Ser Gly As - #n Met Thr Arg Gly Trp         #        45                                                                   - Leu Gln Asp Lys Pro Asn Val Asp Ser Leu Ty - #r Phe Thr Met Phe Asn         #     60                                                                      - Cys Asn Lys Arg Ser Ile Glu Leu Asp Met Ly - #s Thr Pro Glu Gly Lys         # 80                                                                          - Glu Leu Leu Glu Gln Met Ile Lys Lys Ala As - #p Val Met Val Glu Asn         #                 95                                                          - Phe Gly Pro Gly Ala Leu Asp Arg Met Gly Ph - #e Thr Trp Glu Tyr Ile         #           110                                                               - Gln Glu Leu Asn Pro Arg Val Ile Leu Ala Se - #r Val Lys Gly Tyr Ala         #       125                                                                   - Glu Gly His Ala Asn Glu His Leu Lys Val Ty - #r Glu Asn Val Ala Gln         #   140                                                                       - Cys Ser Gly Gly Ala Ala Ala Thr Thr Gly Ph - #e Trp Asp Gly Pro Pro         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Val Ser Gly Ala Ala Leu Gly Asp Ser As - #n Ser Gly Met His Leu         #               175                                                           - Met Ile Gly Ile Leu Ala Ala Leu Glu Met Ar - #g His Lys Thr Gly Arg         #           190                                                               - Gly Gln Lys Val Ala Val Ala Met Gln Asp Al - #a Val Leu Asn Leu Val         #       205                                                                   - Arg Ile Lys Leu Arg Asp Gln Gln Arg Leu Gl - #u Arg Thr Gly Ile Leu         #   220                                                                       - Ala Glu Tyr Pro Gln Ala Gln Pro Asn Phe Al - #a Phe Asp Arg Asp Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asn Pro Leu Ser Phe Asn Asn Ile Thr Ser Va - #l Pro Arg Gly Gly Asn         #               255                                                           - Ala Gly Gly Gly Gly Glu Pro Gly Trp Met Le - #u Lys Cys Lys Gly Trp         #           270                                                               - Glu Thr Asp Ala Asp Ser Tyr Val Tyr Phe Th - #r Ile Ala Ala Asn Met         #       285                                                                   - Trp Pro Gln Ile Cys Asn Met Ile Asp Lys Pr - #o Glu Trp Lys Asp Asp         #   300                                                                       - Pro Ala Tyr Asn Thr Phe Glu Gly Arg Val As - #p Lys Leu Met Asp Ile         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Phe Ser Phe Ile Glu Thr Lys Phe Ala Asp Ly - #s Asp Lys Phe Glu Val         #               335                                                           - Thr Glu Trp Ala Ala Gln Tyr Gly Ile Pro Cy - #s Gly Pro Val Met Ser         #           350                                                               - Met Lys Glu Leu Ala His Asp Pro Ser Leu Gl - #n Lys Val Gly Thr Val         #       365                                                                   - Val Glu Val Val Asp Glu Ile Arg Gly Asn Hi - #s Leu Thr Val Gly Ala         #   380                                                                       - Pro Phe Lys Phe Ser Gly Phe Gln Pro Glu Il - #e Thr Arg Ala Pro Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Leu Gly Glu His Thr Asp Glu Val Leu Lys Gl - #u Leu Gly Leu Asp Asp         #               415                                                           - Ala Lys Ile Lys Glu Leu His Ala Lys Gln Va - #l Val                         #           425                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2088 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATTTGTTTAA ATTGACCTGA ATCAATATTG CCGGATTGAT CTAGGTCAAT GA - #ATGCAAAT         60                                                                          - TGACTTATGT CAATGGTGCC AAATTGACCT AGGTCAACGG GATTTTTAAA GG - #GTATGCGG        120                                                                          - CATACTCGGA ATTGACGTTA AACAACGTTT ATCAAAACCA ACCAAAGAAA GG - #TATTACTC        180                                                                          - ATGAGTAACG ACGACAATGT AGAGTTGACT GATGGCTTTC ATGTTTTGAT CG - #ATGCCCTG        240                                                                          - AAAATGAATG ACATCGATAC CATGTATGGT GTTGTCGGCA TTCCTATCAC GA - #ACCTGGCT        300                                                                          - CGTATGTGGC AAGATGACGG TCAGCGTTTT TACAGCTTCC GTCACGAACA AC - #ACGCAGGT        360                                                                          - TATGCAGCTT CTATCGCCGG TTACATCGAA GGAAAACCTG GCGTTTGCTT GA - #CCGTTTCC        420                                                                          - GCCCCTGGCT TCCTGAACGG CGTGACTTCC CTGGCTCATG CAACCACCAA CT - #GCTTCCCA        480                                                                          - ATGATCCTGT TGAGCGGTTC CAGTGAACGT GAAATCGTCG ATTTCCAAGA CG - #GCGATTAC        540                                                                          - GAAGAAATGG ATCAGATGAA TGTTGCACGT CCACACTGCA AAGCTTCTTT CC - #GTATCAAC        600                                                                          - AGCATCAAAG ACATTCCAAT CGGTATCGCT CGTGCAGTTC GCACCGCTGT AT - #CCGGACGT        660                                                                          - CCAGGTGGTG TTTACGTTGA CTTCCCAGCA AAACTGTTCG GTCAGACCAT TT - #CTGTAGAA        720                                                                          - GAAGCTAACA AACTGCTCTT CAAACCAATC GATCCAGCTC CGGCACAGAT TC - #TTGCTGAA        780                                                                          - GACGCTATCG CTCGCGCTGC TGACCTGATC AAGAACGCCA AACGTCCAGT TA - #TCATGCTG        840                                                                          - GGTAAAGGCG CTGCATACGC ACAATGCGAC GACGAAATCC GCGCACTGGT TG - #AAGAAACC        900                                                                          - GGCATCCCAT TCCTGCCAAT GGGTATGGCT AAAGGCCTGC TGCCTGACAA CC - #ATCCACAA        960                                                                          - TCCGCTGCTG CAACCCGTGC TTTCGCACTG GCACAGTGTG ACGTTTGCGT AC - #TGATCGGC       1020                                                                          - GCTCGTCTGA ACTGGCTGAT GCAGCACGGT AAAGGCAAAA CCTGGGGCGA CG - #AACTGAAG       1080                                                                          - AAATACGTTC AGATCGACAT CCAGGCTAAC GAAATGGACA GCAACCAGCC TA - #TCGCTGCA       1140                                                                          - CCAGTTGTTG GTGACATCAA GTCCGCCGTT TCCCTGCTCC GCAAAGCACT GA - #AAGGCGCT       1200                                                                          - CCAAAAGCTG ACGCTGAATG GACCGGCGCT CTGAAAGCCA AAGTTGACGG CA - #ACAAAGCC       1260                                                                          - AAACTGGCTG GCAAGATGAC TGCCGAAACC CCATCCGGAA TGATGAACTA CT - #CCAATTCC       1320                                                                          - CTGGGCGTTG TTCGTGACTT CATGCTGGCA AATCCGGATA TTTCCCTGGT TA - #ACGAAGGC       1380                                                                          - GCTAATGCAC TCGACAACAC TCGTATGATT GTTGACATGC TGAAACCACG CA - #AACGTCTT       1440                                                                          - GACTCCGGTA CCTGGGGTGT TATGGGTATT GGTATGGGCT ACTGCGTTGC TG - #CAGCTGCT       1500                                                                          - GTTACCGGCA AACCGGTTAT CGCTGTTGAA GGCGATAGCG CATTCGGTTT CT - #CCGGTATG       1560                                                                          - GAACTGGAAA CCATCTGCCG TTACAACCTG CCAGTTACCG TTATCATCAT GA - #ACAATGGT       1620                                                                          - GGTATCTATA AAGGTAACGA AGCAGATCCA CAACCAGGCG TTATCTCCTG TA - #CCCGTCTG       1680                                                                          - ACCCGTGGTC GTTACGACAT GATGATGGAA GCATTTGGCG GTAAAGGTTA TG - #TTGCCAAT       1740                                                                          - ACTCCAGCAG AACTGAAAGC TGCTCTGGAA GAAGCTGTTG CTTCCGGCAA AC - #CATGCCTG       1800                                                                          - ATCAACGCGA TGATCGATCC AGACGCTGGT GTCGAATCTG GCCGTATCAA GA - #GCCTGAAC       1860                                                                          - GTTGTAAGTA AAGTTGGCAA GAAATAATTA GCCCAACTTT GATGACCGGT TA - #CGACCGGT       1920                                                                          - CACATAAAGT GTTCGAATGC CCTTCAAGTT TACTTGAAGG GCATTTTTTT AC - #CTTGCAGT       1980                                                                          - TTATAAACAG GAAAAATTGT ATTCAGAGCG GAAAAGCAGA TTTAAGCCAC GA - #GAAACATT       2040                                                                          #              2088TGCC ATAAACACAT TTTTAAAGCT GGCTTTTT                        - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 568 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ser Asn Asp Asp Asn Val Glu Leu Thr As - #p Gly Phe His Val Leu         #                 15                                                          - Ile Asp Ala Leu Lys Met Asn Asp Ile Asp Th - #r Met Tyr Gly Val Val         #             30                                                              - Gly Ile Pro Ile Thr Asn Leu Ala Arg Met Tr - #p Gln Asp Asp Gly Gln         #         45                                                                  - Arg Phe Tyr Ser Phe Arg His Glu Gln His Al - #a Gly Tyr Ala Ala Ser         #     60                                                                      - Ile Ala Gly Tyr Ile Glu Gly Lys Pro Gly Va - #l Cys Leu Thr Val Ser         # 80                                                                          - Ala Pro Gly Phe Leu Asn Gly Val Thr Ser Le - #u Ala His Ala Thr Thr         #                 95                                                          - Asn Cys Phe Pro Met Ile Leu Leu Ser Gly Se - #r Ser Glu Arg Glu Ile         #           110                                                               - Val Asp Leu Gln Gln Gly Asp Tyr Glu Glu Me - #t Asp Gln Met Asn Val         #       125                                                                   - Ala Arg Pro His Cys Lys Ala Ser Phe Arg Il - #e Asn Ser Ile Lys Asp         #   140                                                                       - Ile Pro Ile Gly Ile Ala Arg Ala Val Arg Th - #r Ala Val Ser Gly Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Pro Gly Gly Val Tyr Val Asp Leu Pro Ala Ly - #s Leu Phe Gly Gln Thr         #               175                                                           - Ile Ser Val Glu Glu Ala Asn Lys Leu Leu Ph - #e Lys Pro Ile Asp Pro         #           190                                                               - Ala Pro Ala Gln Ile Pro Ala Glu Asp Ala Il - #e Ala Arg Ala Ala Asp         #       205                                                                   - Leu Ile Lys Asn Ala Lys Arg Pro Val Ile Me - #t Leu Gly Lys Gly Ala         #   220                                                                       - Ala Tyr Ala Gln Cys Asp Asp Glu Ile Arg Al - #a Leu Val Glu Glu Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Ile Pro Phe Leu Pro Met Gly Met Ala Ly - #s Gly Leu Leu Pro Asp         #               255                                                           - Asn His Pro Gln Ser Ala Ala Ala Thr Arg Al - #a Phe Ala Leu Ala Gln         #           270                                                               - Cys Asp Val Cys Val Leu Ile Gly Ala Arg Le - #u Asn Trp Leu Met Gln         #       285                                                                   - His Gly Lys Gly Lys Thr Trp Gly Asp Glu Le - #u Lys Lys Tyr Val Gln         #   300                                                                       - Ile Asp Ile Gln Ala Asn Glu Met Asp Ser As - #n Gln Pro Ile Ala Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Val Val Gly Asp Ile Lys Ser Ala Val Se - #r Leu Leu Arg Lys Ala         #               335                                                           - Leu Lys Gly Ala Pro Lys Ala Asp Ala Glu Tr - #p Thr Gly Ala Leu Lys         #           350                                                               - Ala Lys Val Asp Gly Asn Lys Ala Lys Leu Al - #a Gly Lys Met Thr Ala         #       365                                                                   - Glu Thr Pro Ser Gly Met Met Asn Tyr Ser As - #n Ser Leu Gly Val Val         #   380                                                                       - Arg Asp Phe Met Leu Ala Asn Pro Asp Ile Se - #r Leu Val Asn Glu Gly         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ala Asn Ala Leu Asp Asn Thr Arg Met Ile Va - #l Asp Met Leu Lys Pro         #               415                                                           - Arg Lys Arg Leu Asp Ser Gly Thr Trp Gly Va - #l Met Gly Ile Gly Met         #           430                                                               - Gly Tyr Cys Val Ala Ala Ala Ala Val Thr Gl - #y Lys Pro Val Ile Ala         #       445                                                                   - Val Glu Gly Asp Ser Ala Phe Gly Phe Ser Gl - #y Met Glu Leu Glu Thr         #   460                                                                       - Ile Cys Arg Tyr Asn Leu Pro Val Thr Val Il - #e Ile Met Asn Asn Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Gly Ile Tyr Lys Gly Asn Glu Ala Asp Pro Gl - #n Pro Gly Val Ile Ser         #               495                                                           - Cys Thr Arg Leu Thr Arg Gly Arg Tyr Asp Me - #t Met Met Glu Ala Phe         #           510                                                               - Gly Gly Lys Gly Tyr Val Ala Asn Thr Pro Al - #a Glu Leu Lys Ala Ala         #       525                                                                   - Leu Glu Glu Ala Val Ala Ser Gly Lys Pro Cy - #s Leu Ile Asn Ala Met         #   540                                                                       - Ile Asp Pro Asp Ala Gly Val Gly Ser Gly Ar - #g Ile Lys Ser Leu Asn         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Val Val Ser Lys Val Gly Lys Lys                                                             565                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #                23TCGA ACG                                                   - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #          31      CTGA TGGCTTTCAT G                                          - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #  18              TC                                                         - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #   17             C                                                          __________________________________________________________________________

We claim:
 1. A process for isolating a formyl-CoA transferasepolypeptide of Oxalobacter formigenes, said method comprising expressinga recombinant polynucleotide molecule encoding said formyl-CoAtransferase polypeptide in a host cell; and isolating said formyl-CoAtransferase polypeptide from said host cell.
 2. The process according toclaim 1, wherein said formyl-CoA transferase polypeptide comprises theamino acid sequence shown in SEQ ID NO.
 2. 3. The process according toclaim 1, wherein said polynucleotide molecule comprises the nucleotidesequence shown in SEQ ID NO. 1.